Early Region 1A Research Articles

Role of early genes in pathogenesis of adenovirus pneumonia.

Intranasal inoculation of type 5 adenovirus into the cotton rat Sigmodon hispidus produces a pneumonia pathologically similar to that in humans, and it, therefore, provides an excellent animal model to investigate the pathogenesis of this disease. The goal of this study was to test the hypothesis that accumulation of viral structural proteins is responsible for a major portion of the cell-damage-producing disease. Since viral DNA replication is essential for synthesis of the viral structural proteins, which are products of late genes, the hypothesis was tested using mutants defective in genes required for DNA synthesis. Most experiments were done with the conditionally lethal temperature-sensitive (ts) mutant H5ts125, which contains a mutation in the early region 2A (E2A) gene encoding the DNA-binding protein. The data show that infection with 1 x 10(9.0) plaque-forming units of H5ts125 induced a pneumonia that was as extensive and qualitatively the same as that after wild-type adenovirus type 5 infection, although H5ts125 did not replicate to produce infectious virus. When cotton rats were infected with 1 x 10(8.0) plaque-forming units of wild-type adenovirus type 5 or H5ts125, the pneumonias that followed were pathologically similar; in the latter phases, however, wild-type virus produced slightly more extensive pneumonia than did H5ts125, probably because its replication permitted infection of more susceptible cells.

Transcriptional repression of the neu protooncogene by the adenovirus 5 E1A gene products.

Amplification/overexpression of the human neu protooncogene has been frequently found in human primary breast and ovarian cancers and is correlated with the number of axillary lymph nodes positive for metastasis in breast cancer patients. Identification of the factors controlling transcription of the neu gene is essential for understanding the mechanisms of neu gene regulation and its role in tumorigenicity. The adenovirus early region 1A (E1A) gene products are pleiotropic transcription regulators of viral and cellular genes and have been identified as a viral suppressor gene for metastasis. Here we demonstrate that transcription of neu can be strongly repressed by the E1A gene products. The 13S and 12S products of E1A gene are effective at repressing neu transcription and the transcriptional repression requires the conserved region 2 of the E1A proteins. The target for E1A repression was localized within a 139-base-pair DNA fragment in the upstream region of the neu promoter. In addition, competition experiments suggest that the sequence TGGAATG, within the 139-base-pair fragment, is an important element for the E1A-induced repression. These results indicate that E1A negatively regulates neu gene expression at the transcriptional level by means of a specific DNA element.

Definition of adenovirus type 5 functions involved in the induction of chromosomal aberrations in human cells

Infection of human embryonic kidney cells with adenovirus type 5 (Ad5) induces aberrations (gaps and breaks) in the cell chromosomes. We have conducted a study utilizing a large number of Ad5 mutants to identify the viral functions that are responsible for the occurrence of cytogenetic damage. The results of our investigation have indicated that expression of the gene products of the Ad5 early region 1A (E1A) is necessary for the induction of chromosomal aberrations and that other early viral gene products do not appear to contribute to this phenotype. We have also shown that expression of both the major E1A gene products, the 243 amino acid and the 289 amino acid proteins, is required for induction of damage at wild-type levels, although the 289 amino acid protein appears to retain detectable activity on its own. Lastly, we have observed that deletions in the amino-terminal region of the E1A proteins and in the transactivating domain of the 289 amino acid protein prevent the occurrence of cytogenetic damage, whereas mutations elsewhere in the proteins do not affect this process.

Cis effect of the type 5 adenovirus E1A gene enhancer element on cellular transformation

Mutants of type 5 adenovirus that lack all or part of the early region 1A (E1A) gene enhancer element transform rodent embryo fibroblast (CREF) cells at higher efficiencies than wild-type virus. An analysis of viral E1A cytoplasmic mRNA levels in mutant and wild-type virus-infected CREF cells revealed no differences in the levels of the E1A mRNAs. This implies that a decrease in the rate of viral E1A gene expression was not responsible for the transforming properties of the enhancer-less viruses. Unlike wild-type virus, however, the mutant viruses were able to replicate their genomes in the normally nonpermissive CREF cells. This change in viral DNA template concentration further resulted in an increase in early gene mRNA concentrations in mutant-virus-infected CREF cells. These studies suggest several possible mechanisms that could be responsible for the increased transforming potentials of these viruses, including 1) a cis effect of removing the viral E1A enhancer element on the efficiency of viral DNA integration, 2) viral DNA replication, or 3) an increase in the levels of the viral E1A and E1B mRNAs owing to viral DNA replication in the virus-infected CREF cells.

Novel phenotype of C3H 10T1/2 fibroblasts cotransfected with the c‐Ha‐ras and adenovirus 5 E1A oncogenes

C3H 10T1/2 fibroblasts are converted to fully transformed phenotype following coexpression of an activated c-Ha-ras gene and either a constitutively expressed viral or cellular myc gene. In this report, we examined whether the early region 1A (E1A) of adenovirus 5, which synergizes with ras to convert primary embryonic cells to a transformed phenotype, can synergize with ras to transform the established mouse embryonic cell line, C3H 10T1/2. We demonstrate that coexpression of ras and E1A generated a transformed phenotype that could be scored by colony assays and by soft agarose assays but not by standard focus assays. The ras-E1A-transformed phenotype relies on sequences present in conserved regions 1 and 2 of the E1A proteins and, in part, on information encoded by the extreme carboxy terminus of E1A. The contrast between the transformed phenotypes generated following the transfection of C3H 10T1/2 cells with either ras and myc or ras and E1A suggests that myc and E1A cooperate with ras to transform C3H 10T1/2 cells by mechanisms that can be distinguished using this established cell line as a model system.

Adenovirus E1A products activate the Ig k-chain enhancer in fibroblasts. A possible involvement of the NF-kB binding site.

Proteins encoded by the early region 1A (E1A) regulate transcription of viral and cellular genes. The mechanism of this trans-regulation remains unclear but it is of considerable interest as an example of transcription regulation through cellular intermediates. We have therefore studied the effect of E1A products on the activity of the k-L chain gene enhancer. In our previous studies we have found that the k-chain enhancer is activated by E1A products in fibroblasts. Using in vivo competition experiments we found that the E1A regulatory effect involves trans-acting factors that can be titrated out by the k-chain enhancer sequences. Our transfection experiments suggest that the k3 enhancer fragment, encompassing the NF-kB binding site, is one of the sites through which the adenovirus E1A products exert their transactivation effect. The level of the nuclear factors that bind to the NF-kB binding site increases in fibroblasts that constitutively express E1A products. Understanding the mechanisms through which E1A regulates transcription should help to illuminate cellular mechanisms of gene control in normal and transformed cells.

Restricted replication of human adenovirus type 5 in mouse cell lines

Infection of mouse BALB/c 3T3 cells by adenovirus 5 resulted in at least 1000-fold lowered yields of virus compared to human cells. The molecular basis of this restriction was analysed at the level of viral gene expression. Steady-state levels of viral DNA and RNA were greatly reduced in infected mouse, compared to human cells. Both early region 1A (E1A) and E1B mRNAs were decreased in mouse cells and their protein products were barely detectable by metabolic labelling of infected cells. The E2A-72 kDa protein and the hexon protein were detected by metabolic labelling, and immunocytochemical analysis showed that they were correctly located in nuclei of infected mouse cells. Only a minor proportion of infected mouse 3T3 cells expressed the E2A-72 kDa or hexon proteins. Low yields of virus were obtained by infection of SV40 transformed BALB/c 3T3 cells showing that SV40 does not provide a helper function for adenovirus 5 growth in this cell system

Multiple transcription factor binding sites mediate adenovirus E1A transactivation

We studied the response of simple synthetic promoter regions to transactivation by the adenovirus early region 1A (E1A) protein. Binding sites for one or two host cell transcription factors were substituted for the E1B promoter region in reconstructed virus mutants, and the response to E1A transactivation was assayed during the early phase of infection. We found that a single CREB/ATF binding site resulted in a surprisingly strong promoter which responded to E1A. A CREB/ATF binding site placed upstream of the E1B TATA box behaved much like the wild-type E1B promoter, which is composed of a single Sp1 binding site plus a TATA box. A single E2F binding site resulted in an extremely weak promoter which did not respond to E1A, much like a single Sp1 site. Two E2F sites in an inverted orientation with the same spacing as in the adenovirus type 2 E2 early promoter produced a strong, E1A-responsive promoter. Substitution of the E4 TATA box region for the E1B TATA box region produced a promoter about five times stronger than the wild-type E1B promoter in the absence of E1A. However, the E4 TATA box substitution did not respond significantly to E1A transactivation. These results directly demonstrate that many different transcription factor binding sites, including the E1B TATA box, a CREB/ATF binding site, and two E2F sites, can mediate E1A transactivation. Other transcription factor binding sites cannot mediate an E1A response; these other sites include the E4 TATA box, a single Sp1 binding site, and a single E2F binding site. Implications of these findings for the mechanism of E1A transactivation are discussed.

Restoration of growth potential in paraclones of human keratinocytes by a viral oncogene.

Human diploid keratinocytes may be divided into three clonal types with differing capacities for proliferation. The paraclone, which has the shortest life span, is limited to 15 divisions, after which all the cells undergo programmed terminal differentiation. By means of a retroviral vector, paraclones which have not completed their life span and which consist of not more than a few hundred cells can be transduced at a high frequency with DNA complementary to the 12S transcript of the adenovirus early region 1A gene. Transformation can be detected within a single cultivation by the formation of progressively growing colonies. The transformants appear to have an unlimited growth potential, and they form a disorganized epidermis when they are grafted as an epithelium onto athymic mice. These experiments clearly show that, in order to be transformed by a viral oncogene, the target cell need not be a stem cell.

Analysis of phosphorylation sites in the exon 1 region of E1A proteins of human adenovirus type 5

Early region 1A (E1A) of human adenovirus type 5 (Ad5) produces five mRNAs that encode proteins of 55, 171, 217, 243, and 289 residues. We have shown previously that the major products of 289 and 243 residues are phosphorylated at a minimum of three sites of which one, Ser-89, is located in the amino terminal half of the protein. In the present report we show that these El A proteins are also phosphorylated at a second site in this region located at Ser-96. The 171 and 217 residue ElA species were also tentatively identified and, as predicted, neither contained the Ser-89 or Ser-96 sites but both appeared to be phosphorylated at the same sites as 289R and 243R toward the carboxy terminus. Studies with mutants in which Ser-89 or Ser-96 were converted to alanine residues indicated that phosphorylation of Ser-89 but not Ser-96 induces the major shift in gel mobility of El A products. However, neither site appears to be of major importance in the regulation of El A-mediated transactivation or transformation.

Phosphorylation at serine 89 induces a shift in gel mobility but has little effect on the function of adenovirus type 5 E1A proteins

Phosphorylation at serine 89 was shown to be the major cause of the shift in gel migration of the 289R and 243R early region 1A (E1A) proteins of human adenovirus type 5. However, conversion of Ser-89 to alanine by site-directed mutagenesis did not abolish E1A transactivating or transforming activities, suggesting that phosphorylation at this site is not necessary for these E1A functions.

Cyclic AMP induction of early adenovirus promoters involves sequences required for E1A trans-activation.

Early in adenovirus infection, the E1A (early region 1A) oncogene products trans-activate the other early viral transcription units, as well as some cellular promoters. The mechanism by which E1A elicits its activity is still unknown. In this report, I show that the adenovirus E2a and E3 promoters are cAMP inducible in rat pheochromocytoma PC12 cells and that this activation requires the presence of the cAMP-dependent protein kinase II. Using deletion mutants of the E2a promoter, it was found that the sequence TACGTCAT located between positions -70 and -77 is involved in both the cAMP response and the E1A trans-activation. Also, in the mutant PC12 cell line A126-2B, which lacks the cAMP-dependent protein kinase II, E1A is still able to activate E2a and E3 promoters. This suggests that E1A products may circumvent the lack of the kinase by activating an alternative signal transduction pathway, which could mimic the effect of agonists of adenylate cyclase. I propose that E1A is capable of modifying by phosphorylation, either directly or indirectly, the transcription factor that binds the ACGTCA motif. Such a factor, termed ATF (adenovirus transcription factor), has already been characterized and appears to have strong similarities to the transcriptional factor CREB (cAMP responsive element binding protein), which binds homologous sequences in cAMP responsive genes, such as somatostatin and c-fos.

Mapping of Cellular Protein-Binding Sites on the Products of Early-Region 1A of Human Adenovirus Type 5

The binding sites for the 300-, 107-, and 105-kilodalton cellular proteins which associate with human adenovirus type 5 E1A products were studied with E1A deletion mutants. All appeared to bind to the amino-terminal half of E1A products in regions necessary for oncogenic transformation. These results suggest that these cellular species may be important for the biological activity of E1A products.

Mapping of cellular protein-binding sites on the products of early-region 1A of human adenovirus type 5.

The binding sites for the 300-, 107-, and 105-kilodalton cellular proteins which associate with human adenovirus type 5 E1A products were studied with E1A deletion mutants. All appeared to bind to the amino-terminal half of E1A products in regions necessary for oncogenic transformation. These results suggest that these cellular species may be important for the biological activity of E1A products.

Identification of the phosphorylation sites in early region 1A proteins of adenovirus type 5 by amino acid sequencing of peptide fragments.

We have mapped the positions of three of the phosphorylation sites on the 289 and 243 residue (289R and 243R) early region 1A (E1A) proteins of human adenovirus type 5 (Ad5). These proteins, which play roles in both transcriptional control and oncogenic transformation, have identical sequences except for the presence in 289R of 46 additional internal amino acids. Phosphorylation was detected exclusively at serine residues. E1A proteins purified from [35S]methionine- or [32P]orthophosphate-labeled Ad5-infected cells were digested with trypsin, and two phosphopeptides were isolated by reverse-phase chromatography and subjected to automated Edman degradation. The major species was shown to contain a single phosphorylation site at Ser-219. The second phosphopeptide was shown to contain at least one phosphorylation site at Ser-231. A third phosphorylated tryptic peptide could not be eluted from the column but was isolated using an E1A-specific rat monoclonal antibody. Following subcleavage by Staphylococcus aureus V-8 protease, this peptide was shown to contain at least one phosphorylation site at Ser-89. The present data indicate that both the 289R and 243R E1A proteins are phosphorylated at the same sites, at least one in the amino terminal half of the molecule, and at least two toward the carboxyl terminus.

Physical organization of the enteric adenovirus type 41 early region 1A

Enteric adenovirus types 40 and 41 (Ad40 and Ad41), representing subgenus F, differ from all other human adenoviruses by being so fastidious that productive replication does not occur in conventional established cell lines. They are dependent of the Ad5 early regions ElA and ElB since they can not grow in HEK cells, only in 293 HEK cells transformed by Ad5 E1. The overall genetic organization of Ad41 ElA is similar to the E1A region of other characterized human adenoviruses but it is slightly shorter, comprising 1350 bp. The inverted terminal repeat (ITR) at the 5′ end of both Ad40 and Ad41 consists of 163 nucleotides, being similar to the ITR of Ad12 (subgenus A) and longer than the ITRs of adenoviruses of subgenera B, C, and E. The early mRNA products (12 and 13 S) can be translated into a 222-amino acid (aa) and a 251-aa tentative protein, respectively. In a comparison of the Ad41 251-aa protein with corresponding peptides of Ad12, Ad7, Ad5, and Ad4, three conserved amino acid sequences CS1-CS3 can be found. In the second conserved domain CS2, which is particularly acidic, the homology is very high within all five serotypes compared. Only one among eight conserved amino acids differs in the Ad41 251-aa protein. Within CS1 and CS3 which exhibit a hydrophilic and a hydrophobic character, respectively, the amino acid composition of the Ad41 protein is less conserved than the corresponding regions in all other analyzed adenovirus types. Ten of 16 conserved amino acids in CS1 are shared by Ad41 and 18 of 23 conserved amino acids in CS3 are shared by Ad41.

Regulation of the Ig kappa-chain enhancer by the adenovirus E1A gene products. Repression in lymphoid cells, activation in fibroblasts.

Proteins encoded by early region 1A (E1A) regulate transcription of viral and cellular genes. The mechanism of this trans-activation is not understood, but is of considerable interest as an example of transcription regulation through a cellular intermediate. We have therefore studied the effect of E1A products on the activity of the kappa L chain gene promoter and enhancer. By using transient and stable transfections into lymphoid and nonlymphoid cells, we found that the E1A proteins have a pleiotropic effect on the regulation of the mouse kappa-chain gene enhancer. In lymphoid cells the E1A products repress kappa-chain enhancer, whereas in fibroblasts, the kappa-chain enhancer is activated by the E1A products whether the E1A gene is in an extrachromosomal location or stably integrated in the genome. Furthermore, a functional kappa-chain promoter, containing the octanucleotide and "TATA" sequences is needed in order to be transcribed in E1A-producing cells. This ability of E1A products to negatively and positively regulate kappa-chain transcription may reflect a more general phenomenon in which a given cellular protein could participate in a variety of different cellular controls.

Two regions of the adenovirus early region 1A proteins are required for transformation.

Regions of the adenovirus type 5 early region 1A (E1A) proteins that are required for transformation were defined by using a series of deletion mutants. Deletion mutations collectively spanning the entire protein-coding region of E1A were constructed and assayed for their ability to cooperate with an activated ras oncogene to induce transformation in primary baby rat kidney cells. Two regions of E1A (amino acids 1 to 85 and 121 to 127) were found to be essential for transformation. Deletion of all or part of the region from amino acids 121 to 127 resulted in a total loss of transforming ability. An adjacent stretch of amino acids (residues 128 to 139), largely consisting of acidic residues, was found to be dispensable for transformation but appeared to influence the efficiency of transformation. Amino acids 1 to 85 made up a second region of the E1A protein that was essential for transformation. Deletion of all or part of this region resulted in a loss of the transforming activity. Even a mutation resulting in a single amino acid change at position 2 of the polypeptide chain was sufficient to eliminate transformation. Deletion of amino acids 86 to 120 or 128 to 289 did not eliminate transformation, although some mutations in these regions had lowered efficiencies of transformation. Foci induced by transformation-competent mutants could be expanded into cell lines that retained their transformed morphology and constitutively expressed the mutant E1A proteins.

Binding of cellular polypeptides to human adenovirus type 5 E1A proteins produced in Escherichia coli

Cellular proteins of 300, 107, 105, 68 and 65 kDa have previously been shown to associate specifically with the early region 1A (E1A) proteins of human adenovirus type 5. In the present study we report that, to varying degrees, these proteins also were capable of binding to E1A products produced in Escherichia coli from plasmids carrying cDNAs corresponding to the 1.1- and 0.9-kb E1A mRNAs. When these purified E1A proteins were mixed in solution with extracts from mock-infected human cells, the 68- and 65-kDa species bound very efficiently to the 1.1-kb mRNA product and somewhat less so with that of the 0.9-kb mRNA. The 107-, 105-, and 300-kDa species bound poorly, if at all, to both E1A products. Using the E1A 1.1-kb mRNA product which had been covalently attached to Sepharose beads, the 68-, 65-, and 300-kDa species bound efficiently, and binding of protein which migrated in SIDS gels in the region of the 107- and 105-kDa species was also observed. In addition to these proteins, several other cellular polypeptides of 30, 33, 75, 95, 150, 180, and >300 kDa were shown to bind to E1A-Sepharose and thus may also be E1A-binding proteins. The present data confirm the specificity of the previously identified cellular proteins for E1 A products and show that binding of the 300-, 65-, and 68-kDa species does not require the presence of any other viral polypeptide. In contrast, the inefficient binding of the 107- and 105-kDa species to Escherichia coli-expressed E1A protein may suggest that these interactions require either eukaryotic-specific post-translational modifications of the E1A protein, or the presence of additional Ad5 gene products.

Promoter trans-activation of protooncogenes c-fos and c-myc, but not c-Ha-ras, by products of adenovirus early region 1A.

The E1A (early region 1A) oncogene products of adenovirus type 2 trans-activate the other early viral transcription units, as well as some cellular promoters. Using a short-term cotransfection assay in murine NIH 3T3 fibroblasts, we show that c-fos and c-myc promoter activities are stimulated by the E1A proteins, whereas c-Ha-ras transcription is not affected. The product of E1A 13S mRNA is responsible for the trans-activation, whereas the 12S mRNA product has no effect. Analysis of the c-fos promoter sequences required for the E1A stimulation shows that responsive sequences are located between positions -402 and -240 upstream of the transcription initiation site. This same region also contains the c-fos serum-responsive element. Furthermore, transcription of the endogenous c-fos gene in HeLa cells is increased after E1A transfection.